Isolation and characterization of the compounds responsible for the antimutagenic activity of Combretum microphyllum (Combretaceae) leaf extracts

Abstract:

BACKGROUND : Mutations play a major role in the pathogenesis and development of several chronic degenerative
diseases including cancer. It follows, therefore that antimutagenic compound may inhibit the pathological process
resulting from exposure to mutagens. Investigation of the antimutagenic potential of traditional medicinal plants
and compounds isolated from plant extracts provides one of the tools that can be used to identify compounds
with potential cancer chemopreventive properties. The aim of this study was to isolate and characterise the
compounds responsible for the antimutagenic activity of Combretum microphyllum.
METHODS : The methanol leaf extract of C. microphyllum was evaluated for antimutagenicity in the Ames/microsome
assay using Salmonella typhimurium TA98. TA100 and TA102. Solvent-solvent fractionation was used to partition the
extracts and by using bioassay-guided fractionation, three compounds were isolated. The antimutagenic activity of the
three compounds were determined in the Ames test using Salmonella typhimurium TA98, TA100 and TA102. The
antioxidant activity of the three compounds were determined by the quantitative 2,2-diphenyl-1-picrylhydrazyl (DPPH)-
free radical scavenging method. The cytotoxicity was determined in the MTT assay using human hepatocytes.
RESULTS : A bioassay-guided fractionation of the crude extracts for antimutagenic activity led to the isolation of three
compounds; n-tetracosanol, eicosanoic acid and arjunolic acid. Arjunolic acid was the most active in all three tested
strains with a antimutagenicity of 42 ± 9.6%, 36 ± 1.5% and 44 ± 0.18% in S. typhimurium TA98, TA100 and TA102
respectively at the highest concentration (500 μg/ml) tested, followed by eicosanoic acid and n-tetracosanol. The
antioxidant activity of the compounds were determined using the quantitative 2,2 diphenyl-1-picryhydrazyl (DPPH)-free
radical scavenging method. Only arjunolic acid had pronounced antioxidant activity (measured as DPPH-free scavenging
activity) with an EC50 value of 0.51 μg/ml. The cytotoxicity of the isolated compounds were determined in the MTT assay
using human hepatocytes. The compounds had low cytotoxicity at the highest concentration tested with LC50 values
>200 μg/ml for n-tetracosanol and eicosanoic acid and 106.39 μg/ml for arjunolic acid.
CONCLUSIONS : Based on findings from this study, compounds in leaf extracts of C. microphyllum protected against 4-
NQO and MMC induced mutations as evident in the Ames test. The antimutagenic activity of arjunolic acid may, at
least in part, be attributed to its antioxidant activity resulting in the detoxification of reactive oxygen species produced
during mutagenesis.